U.S. patent application number 12/514244 was filed with the patent office on 2010-02-18 for caspase inhibitors based on pyridazinone scaffold.
This patent application is currently assigned to LG Life Sciences Ltd.. Invention is credited to Hye Kyung Chang, Chul Woong Chung, Yong Jin Jang, Sung Sub Kim, Kyeong Sik Min, Yeong Soo Oh, Jung Gyu Park, Mi Jeong Park.
Application Number | 20100041661 12/514244 |
Document ID | / |
Family ID | 39364683 |
Filed Date | 2010-02-18 |
United States Patent
Application |
20100041661 |
Kind Code |
A1 |
Chang; Hye Kyung ; et
al. |
February 18, 2010 |
CASPASE INHIBITORS BASED ON PYRIDAZINONE SCAFFOLD
Abstract
The present invention relates to a pyridazinone derivative which
can be used as a caspase inhibitor, process for the preparation
thereof, and pharmaceutical composition for inhibiting caspase
comprising the same.
Inventors: |
Chang; Hye Kyung; (Daejeon,
KR) ; Oh; Yeong Soo; (Daejeon, KR) ; Jang;
Yong Jin; (Daejeon, KR) ; Kim; Sung Sub;
(Daejeon, KR) ; Min; Kyeong Sik; (Daejeon, KR)
; Chung; Chul Woong; (Daejeon, KR) ; Park; Mi
Jeong; (Daejeon, KR) ; Park; Jung Gyu;
(Daejeon, KR) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
LG Life Sciences Ltd.
Seoul
KR
|
Family ID: |
39364683 |
Appl. No.: |
12/514244 |
Filed: |
October 26, 2007 |
PCT Filed: |
October 26, 2007 |
PCT NO: |
PCT/KR2007/005303 |
371 Date: |
June 4, 2009 |
Current U.S.
Class: |
514/247 ;
544/239 |
Current CPC
Class: |
A61P 31/04 20180101;
A61P 25/28 20180101; A61P 29/00 20180101; A61P 1/16 20180101; A61P
37/06 20180101; A61P 43/00 20180101; C07D 237/14 20130101; A61P
3/10 20180101; A61P 31/18 20180101; A61P 9/10 20180101; A61P 9/00
20180101; A61P 25/00 20180101; A61P 1/04 20180101 |
Class at
Publication: |
514/247 ;
544/239 |
International
Class: |
C07D 237/00 20060101
C07D237/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 9, 2006 |
KR |
10-2006-0110501 |
Claims
1. A compound of formula (1): ##STR00015## in which I) R.sup.1
represents H, C.sub.1-C.sub.5-alkyl, C.sub.3-C.sub.10-cycloalkyl,
aryl, or a side chain residue of all the natural amino acids, II)
R.sup.2 represents H, C.sub.1-C.sub.5-alkyl,
C.sub.3-C.sub.10-cycloalkyl, aryl, or a side chain residue of all
the natural amino acids, III) R.sup.3 represents H,
C.sub.1-C.sub.5-alkyl, aryl, hydroxy, C.sub.1-C.sub.5-alkoxy, or
halogen, IV) R.sup.4 represents H, C.sub.1-C.sub.5-alkyl,
C.sub.3-C.sub.10-cycloalkyl, or aryl, V) R.sup.5 represents H,
C.sub.1-C.sub.5-alkyl, C.sub.3-C.sub.10-cycloalkyl, or aryl, VI)
R.sup.6 and R.sup.7 independently of one another each represent H,
C.sub.1-C.sub.5-alkyl, C.sub.3-C.sub.10-cycloalkyl, or aryl, VII) X
represents --CH.sub.2OR.sup.9 (R.sup.9 is C.sub.1-C.sub.5-alkyl,
C.sub.3-C.sub.10-cycloalkyl, or aryl), --CH.sub.2C(.dbd.O)R.sup.10
(R.sup.10 is C.sub.1-C.sub.5-alkyl, C.sub.3-C.sub.10-cycloalkyl, or
aryl), or --CH.sub.2--W (W is halogen), or pharmaceutically
acceptable salt thereof.
2. The compound of claim 1 wherein R.sup.5 represents
C.sub.1-C.sub.5-alkyl substituted by C.sub.3-C.sub.10-cycloalkyl or
aryl, each of which is substituted or unsubstituted; or represents
substituted or unsubstituted aryl, or pharmaceutically acceptable
salt thereof.
3. The compound of claim 2 wherein R.sup.5 represents
C.sub.1-C.sub.5-alkyl substituted by C.sub.3-C.sub.10-cycloalkyl or
aryl, each of which is unsubstituted or substituted by one or more
substituents selected from the group consisting of
C.sub.1-C.sub.5-alkyl, hydroxy, C.sub.1-C.sub.5-alkoxy and halogen;
or represents aryl which is unsubstituted or substituted by one or
more substituents selected from the group consisting of
C.sub.1-C.sub.5-alkyl, hydroxy, C.sub.1-C.sub.5-alkoxy and halogen,
or pharmaceutically acceptable salt thereof.
4. The compound of claim 1 wherein I) R.sup.1 represents a side
chain residue of all the natural amino acids, II) R.sup.2
represents C.sub.1-C.sub.5-alkyl, III) R.sup.3 represents H,
C.sub.1-C.sub.5-alkyl, aryl, C.sub.1-C.sub.5-alkoxy, or halogen,
IV) R.sup.4 represents H, V) R.sup.5 represents
C.sub.1-C.sub.5-alkyl substituted by C.sub.3-C.sub.10-cycloalkyl or
aryl, each of which is unsubstituted or substituted by one or more
substituents selected from the group consisting of
C.sub.1-C.sub.5-alkyl, hydroxy, C.sub.1-C.sub.5-alkoxy and halogen;
or represents aryl which is unsubstituted or substituted by one or
more substituents selected from the group consisting of
C.sub.1-C.sub.5-alkyl, hydroxy, C.sub.1-C.sub.5-alkoxy and halogen,
VI) R.sup.6 and R.sup.7 independently of one another each represent
H, VII) X represents --CH.sub.2OR.sup.9 (R.sup.9 is
C.sub.1-C.sub.5-alkyl, C.sub.3-C.sub.10-cycloalkyl, or aryl),
--CH.sub.2C(.dbd.O)R.sup.10 (R.sup.10 is C.sub.1-C.sub.5-alkyl,
C.sub.3-C.sub.10-cycloalkyl, or aryl), or --CH.sub.2--W (W is
halogen), or pharmaceutically acceptable salt thereof.
5. The compound of claim 1 wherein I) R.sup.1 represents
--CH.sub.2COOH, II) R.sup.2 represents C.sub.1-C.sub.5-alkyl, III)
R.sup.3 represents H, C.sub.1-C.sub.5-alkyl, aryl,
C.sub.1-C.sub.5-alkoxy, or halogen, IV) R.sup.4 represents H, V)
R.sup.5 represents C.sub.1-C.sub.5-alkyl substituted by
C.sub.3-C.sub.10-cycloalkyl or aryl, each of which is unsubstituted
or substituted by one or more substituents selected from the group
consisting of C.sub.1-C.sub.5-alkyl, hydroxy,
C.sub.1-C.sub.5-alkoxy and halogen; or represents aryl which is
unsubstituted or substituted by one or more substituents selected
from the group consisting of C.sub.1-C.sub.5-alkyl, hydroxy,
C.sub.1-C.sub.5-alkoxy and halogen, VI) R.sup.6 and R.sup.7
independently of one another each represent H, VII) X represents
--CH.sub.2O-(2,3,5,6-tetrafluorophenyl),
--CH.sub.2O-(2,6-dichlorobenzoyl) or --CH.sub.2--F, or
pharmaceutically acceptable salt thereof.
6.
(S)-3-{2-[5-(2-tert-butyl-benzyl)-6-oxo-6H-pyridazin-1-yl]-butyrylamin-
o}-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid.
7. A pharmaceutical composition for inhibiting caspase, comprising
the compound as defined in claim 1 or pharmaceutically acceptable
salt thereof as an active ingredient together with a
pharmaceutically acceptable carrier.
8. The composition of claim 7 for preventing inflammation and
apoptosis.
9. The composition of claim 7 for the treatment or prevention of
dementia, cerebral stroke, brain impairment due to AIDS, diabetes,
gastric ulcer, cerebral injury by hepatitis, hepatitis-induced
hepatic diseases, acute hepatitis, fulminant hepatic failure,
sepsis, organ transplantation rejection, rheumatic arthritis,
cardiac cell apoptosis due to ischemic cardiac diseases, or liver
cirrhosis.
10. The composition of claim 7 for the treatment of acute hepatitis
or liver cirrhosis.
11. The composition of claim 7 for the treatment of rheumatic
arthritis.
12. A use of the compound as defined in claim 1 or pharmaceutically
acceptable salt thereof for inhibiting caspase.
13. A method for preventing inflammation and apoptosis in a
patient, which comprises administering a therapeutically effective
amount of the compound as defined in claim 1 or pharmaceutically
acceptable salt thereof to the patient.
14. A method for the treatment or prevention of dementia, cerebral
stroke, brain impairment due to AIDS, diabetes, gastric ulcer,
cerebral injury by hepatitis, hepatitis-induced hepatic diseases,
acute hepatitis, fulminant hepatic failure, sepsis, organ
transplantation rejection, rheumatic arthritis, cardiac cell
apoptosis due to ischemic cardiac diseases, or liver cirrhosis in a
patient, which comprises administering a therapeutically effective
amount of the compound as defined in claim 1 or pharmaceutically
acceptable salt thereof to the patient.
15. A pharmaceutical composition for inhibiting caspase, comprising
the compound as defined in claim 6 or pharmaceutically acceptable
salt thereof as an active ingredient together with a
pharmaceutically acceptable carrier.
16. A use of the compound as defined in claim 6 or pharmaceutically
acceptable salt thereof for inhibiting caspase.
17. A method for preventing inflammation and apoptosis in a
patient, which comprises administering a therapeutically effective
amount of the compound as defined in claim 6 or pharmaceutically
acceptable salt thereof to the patient.
18. A method for the treatment or prevention of dementia, cerebral
stroke, brain impairment due to AIDS, diabetes, gastric ulcer,
cerebral injury by hepatitis, hepatitis-induced hepatic diseases,
acute hepatitis, fulminant hepatic failure, sepsis, organ
transplantation rejection, rheumatic arthritis, cardiac cell
apoptosis due to ischemic cardiac diseases, or liver cirrhosis in a
patient, which comprises administering a therapeutically effective
amount of the compound as defined in claim 6 or pharmaceutically
acceptable salt thereof to the patient.
Description
TECHNICAL FIELD
[0001] The present invention relates to a pyridazinone derivative
or pharmaceutically acceptable salt thereof as an inhibitor against
various caspases including caspase-1
[interleukin-1.beta.-converting enzyme, ICE], caspase-3
[apopain/CPP-32], caspase-8, and caspase-9, and a pharmaceutical
composition for the inhibition of caspase comprising the same.
BACKGROUND ART
[0002] Caspase is a new kind of cysteine protease in the form of
.alpha..sub.2.beta..sub.2 tetramer discovered during the last 10
years. About 14 kinds thereof have been known until now.
Caspase-1(ICE), one of them, is a kind of cytokine and participates
in converting the biologically inactive prointerleukin-1.beta. to
the active interleukin-1.beta.. Interleukin-1 consists of
interleukin-1.alpha. and interleukin-1.beta., both of which are
synthesized in monocytes in the form of 31 KDa precursor. Only
prointerleukin-1.beta. is activated by ICE. The positions
hydrolyzed by caspase-1 are Asp.sup.27-Gly.sup.28 and
Asp.sup.116-Ala.sup.117. The hydrolysis of the latter position
gives interleukin-1.beta.. Interleukin-1.beta. has been reported to
act as an important mediator in causing inflammation (1,3).
Caspase-1 has been discovered for the first time in 1989, and the
three dimensional structure thereof was determined by X-ray
crystallographic method by two independent study groups.
[0003] Caspase-3(CPP-32) is broadly studied for its role or
mechanism for action, and its three dimensional structure was
determined in 1996 (2). Caspase-3(apopain) activated from
procaspase-3 is hydrolyzed at the position of
(P.sub.4)Asp-X-X-Asp(P.sub.1) motif, and the known substrates
include poly(ADP-ribose) polymerase, U1 70,000 Mr small nuclear
ribonucleoprotein, catalytic subunit of 460,000 Mr DNA-dependent
protein kinase, etc. The X-ray structure of caspase-7 has been
reported to be very similar to that of caspase-3 (4).
[0004] Caspase-8 and 9 are present in the upstream of
caspase-3,6,7, and all of these caspases are known to participate
in the apoptosis cascade. The X-ray structure of caspase-8 was
determined in 1999 (5), and particularly the inhibitors thereof may
be advantageously used for treating the diseases related to
apoptosis.
[0005] Caspase inhibitors mean these compounds that inhibit the
activity of caspase, and so control such symptoms as inflammation,
apoptosis, etc. caused by the caspase activity. Diseases or
symptoms that may be treated or attenuated by administering the
inhibitors include the following: dementia, cerebral stroke, brain
impairment due to AIDS, diabetes, gastric ulcer, cerebral injury by
hepatitis virus, hepatitis-induced hepatic diseases, acute
hepatitis, fulminant hepatic failure, sepsis, organ transplantation
rejection, rheumatic arthritis, ischemic cardiac diseases, and
liver cirrhosis(6).
[0006] Among the caspase inhibitors known until now, the most noted
irreversible inhibitors are the following:
##STR00001##
[0007] Both the above inhibitors exhibit their activity based on
the common mechanism that they irreversibly inactivate the enzyme
to suppress the cell apoptosis (irreversible, broad-spectrum
inhibitor). It has been reported that irreversible inhibitor has
much more effective inhibitory activity than reversible inhibitor
(7). Both IDN-1965 of IDUN Co. and MX-1013 of Maxim Co. are
reported to show activity in cell apoptosis model for hepatic
injury (8, 9). These compounds are now in the stage of preclinical
test.
[0008] The irreversible inhibitor IDN-6556 is now in the stage of
phase II clinical trial as a hepatoprotective agent for hepatitis C
patients (10, 6-liver cirrhosis-i).
##STR00002##
REFERENCES
[0009] (1) Inflammation: Basic Principles and Clinical Correlates,
2nd ed., ed by Gallin, Goldstein and Snyderman. Raven Press Ltd.,
New York. 1992, pp. 211-232; Blood, 1996, 87(6), 2095-2147. [0010]
(2) Wilson, K. P. et al, Nature, 1994, 370. 270; Walker, N. P. C.
et al. Cell, 1994, 78, 343; Nature Structural Biology, 1996, 3(7),
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[0012] (4) Wei, Y. et al, Chemistry and Biology, 2000, 7, 423.
[0013] (5) Blanchard H. et al, Structure, 1999, 7, 1125; Blanchard
H. et al, J. of Mol. Biol., 2000, 302, 9. [0014] (6) References for
caspase related diseases [0015] Dementia: Arch Neurol 2003 March;
60(3):369-76, Caspase gene expression in the brain as a function of
the clinical progression of Alzheimer disease. Pompl P N, Yemul S,
Xiang Z, Ho L, Haroutunian V, Purohit D, Mohs R, Pasinetti G M.
[0016] Cerebral stroke: Proc Natl Acad Sci USA 2002 Nov. 12;
99(23):15188-93, Caspase activation and neuroprotection in
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vitro oxygen glucose deprivation. Le D A, Wu Y, Huang Z, Matsushita
K, Plesnila N, Augustinack J C, Hyman B T, Yuan J, Kuida K, Flavell
R A, Moskowitz M A. [0017] Brain impairment due to AIDS: J Neurosci
2002 May 15; 22(10):4015-24, Caspase cascades in human
immunodeficiency virus-associated neurodegeneration. Garden G A,
Budd S L, Tsai E, Hanson L, Kaul M, D'Emilia D M, Friedlander R M,
Yuan J, Masliah E, Lipton S A. [0018] Diabetes: Diabetes 2002 June;
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mitochondrial cytochrome C-mediated caspase-3 activation pathway.
Cai L, Li W, Wang G, Guo L, Jiang Y, Kang Y J. [0019] Gastric
ulcer: J Physiol Pharmacol 1998 December; 49(4):489-500, Role of
basic fibroblast growth factor in the suppression of apoptotic
caspase-3 during chronic gastric ulcer healing. Slomiany B L,
Piotrowski J, Slomiany A. [0020] Cerebral injury by hepatitis
virus: J Viral Hepat 2003 March; 10(2):81-6, Cerebral dysfunction
in chronic hepatitis C infection. Forton D M, Taylor-Robinson S D,
Thomas H C. [0021] Fulminant hepatic failure: Gastroenterology 2000
August; 119(2):446-60, Tumor necrosis factor alpha in the
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K, Leifeld L, Grundmann D, Ramakers J, Eckert K, Spengler U,
Brenner D, Manns M, Trautwein C. [0022] Sepsis: Nat Immunol 2000
December; 1(6):496-501, Caspase inhibitors improve survival in
sepsis: a critical role of the lymphocyte. Hotchkiss R S, Chang K
C, Swanson P E, Tinsley K W, Hui J J, Klender P, Xanthoudakis S,
Roy S, Black C, Grimm E, Aspiotis R, Han Y, Nicholson D W, Karl I
E. [0023] Organ transplantation rejection: Xenotransplantation 2001
May; 8(2):115-24, In vitro prevention of cell-mediated xeno-graft
rejection via the Fas/FasL-pathway in CrmA-transducted porcine
kidney cells. Fujino M, Li X K, Suda T, Hashimoto M, Okabe K,
Yaginuma H, Mikoshiba K, Guo L, Okuyama T, Enosawa S, Amemiya H,
Amano T, Suzuki S. [0024] Rheumatic arthritis: Prog Med Chem 2002;
39:1-72, Caspase inhibitors as anti-inflammatory and antiapoptotic
agents. Graczyk P P. [0025] Ischemic cardiac diseases: Am J Physiol
Heart Circ Physiol 2002 September; 283(3):H990-5, Hypoxia-induced
cleavage of caspase-3 and DFF45/ICAD in human failed
cardiomyocytes. Todor A, Shafov V G, Tanhehco E J, Silverman N,
Bernabei A, Sabbah H N. [0026] Anti-inflammation: J Immunol 2003
Mar. 15; 170(6):3386-91, A broad-spectrum caspase inhibitor
attenuates allergic airway inflammation in murine asthma model.
Iwita A, Nishio K, Winn R K, Chi E Y, Henderson W R Jr, Harlan J M.
[0027] Hepatitis-induced hepatic diseases: i) J Viral Hepat. 2003
September; 10(5): 335-42. Apoptosis in hepatitis C Kountouras J,
Zavos C, Chatzopoulos D.; ii) Apoptosis 2003 December; 8(6): 655-63
Apoptosis participates to liver damage in HSV-induced fulminant
hepatitis. Pretet J L, Pelletier L, Bernard B, Coumes-Marquet S,
Kantelip B, Mougin C.; iii) Proc Natl Acad Sci USA. 2003 Jun. 24;
100(13):7797-802. Caspase 8 small interfering RNA prevents acute
liver failure in mice. Zender L, Hutker S, Liedtke C, Tillmann H L,
Zender S, Mundt B, Waltemathe M, Gosling T, Flemming P, Malek N P,
Trautwein C, Manns M P, Kuhnel F, Kubicka S. [0028] Liver
cirrhosis: i) J Pharmacol Exp Ther. 2004 March; 308(3): 1191-6, The
caspase inhibitor Idn-6556 attenuates hepatic injury and fibrosis
in the bile duct ligated mouse. Canbay A., Fledstein A., Baskin-Bey
E., Bronk F. S. Gores G J.; ii) Hepatology. 2004 February; 39 (2):
273-8, Apoptosis: the nexus of liver injury and fibrosis. Canbay A,
Friedman S, Gores G J.; iii) Hepatology. 2003 November; 38(5):
1188-98, Kupffer cell engulfment of apoptotic bodies stimulates
death ligand and cytokine expression. Canbay A, Feldstein A E,
Higuchi H, Werneburg N, Grambihler A, Bronk S F, Gores G J. [0029]
(7) Wu J. et al, Methods: A Companion to Methods in Enzymology,
1999, 17, 320. [0030] (8) Hoglen N. C. et al, J. of Pharmacoloy and
Experimental Therapeutics, 2001, 297, 811. [0031] (9) Jaeschke H.
et al, Toxicology and Applied Pharmacology, 2000, 169, 77. [0032]
(10) Hoglen N. C. et al, J. Pharmacol Exp. Ther., 2004, 309(2):634.
Characterization of IDN-6556
(3-[2-(2-tert-butyl-phenylaminooxalyl)-amino]-propionylamino)-4--
oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid): a
liver-targeted caspase inhibitor.
DISCLOSURE
Technical Problem
[0033] The present inventors have extensively studied to design
novel compounds which can be used as an effective and more
selective inhibitor against caspases.
Technical Solution
[0034] To achieve such a subject, the present inventors synthesized
various compounds, and determined their binding ability and
inhibitory activity for caspases. As a result, the inventors have
discovered that a compound of the following formula (1) does meet
such requirements, and completed the present invention.
##STR00003##
[0035] in which
[0036] R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7 and X are defined below.
[0037] Therefore, the present invention provides the novel
pyridazinone derivative of formula (1) or pharmaceutically
acceptable salt thereof having effective inhibitory activity
against caspases.
[0038] It is another object of the present invention to provide a
pharmaceutical composition for inhibiting caspase, specifically a
composition for preventing inflammation and apoptosis, comprising
the compound of formula (1) or pharmaceutically acceptable salt
thereof as an active ingredient together with the pharmaceutically
acceptable carrier.
ADVANTAGEOUS EFFECTS
[0039] The compound of formula (I) according to the present
invention has an excellent inhibitory activity against caspase, and
so can be advantageously used for the treatment of various diseases
and symptoms mediated by caspase.
BEST MODE
[0040] First of all, the important terms in the present invention
are defined as follows:
[0041] a) C.sub.1-C.sub.5-alkyl: Straight-chain or branched
hydrocarbons having 1 to 5 carbon atoms, that include methyl,
ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, etc., but are
not limited thereto.
[0042] b) C.sub.3-C.sub.10-cycloalkyl: Cyclic hydrocarbons having 3
to 10 carbon atoms, that include cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, etc., but are not limited thereto.
[0043] c) Aryl: Aryl group includes all the aromatic,
heteroaromatic and their partially reduced derivatives. The
aromatic group means a 5 to 15-membered single or fused unsaturated
hydrocarbon. The heteroaromatic group means the aromatic group
containing 1 to 5 hetero atoms selected from a group consisting of
oxygen, sulfur, and nitrogen. The aryl group includes phenyl,
naphthyl, indolyl, quinolinyl, isoquinolyl, imidazolinyl,
isoxazolyl, oxazolyl, thiazolyl, etc., but is not limited
thereto.
[0044] One or more hydrogens in said C.sub.1-C.sub.5-alkyl,
C.sub.3-C.sub.10-cycloalkyl or aryl group may be replaced with a
group(s) selected from the following: acyl, amino, carboalkoxy,
carboxy, carboxyamino, cyano, halo, hydroxy, nitro, thio, alkyl,
cycloalkyl, alkoxy, aryl, aryloxy, sulfoxy, and guanido group.
[0045] d) Natural amino acid includes the following: Glycine,
Alanine, Valine, Leucine, Isoleucine, Serine, Threonine, Cysteine,
Methionine, Proline, Aspartic acid, Asparagine, Glutamic acid,
Glutamine, Lysine, Arginine, Histidine, Phenylalanine, Tyrosine,
and Tryptophan.
[0046] Further, the present specification includes the following
abbreviations:
[0047] N-bromosuccinimide: NBS
[0048]
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumhexafluoroph-
osphate]: HATU
[0049] N,N-dimethyl formamide: DMF
[0050] Dimethylsulfoxide: DMSO
[0051] N-methylmorpholine: NMM
[0052] 2,2'-Azobis(2-methyl propionitrile): AIBN
[0053] 2,2,6,6-Tetramethyl-1-piperidinyloxy, free radical:
TEMPO
[0054] Lithium bis(trimethylsilyl)amide: LiHMDS
[0055] N-(2-Hydroxyethyl)piperazine-N'-(2'-ethanesulfonic acid):
HEPES
[0056] 3-[(3-Cholamidopropyl)dimethylamino]-1-propanesulfonate:
CHAPS
[0057] Ethylenediaminetetraacetic acid: EDTA
[0058] Dithiothreitol: DTT
[0059] The present invention will be explained more in detail
below. One aspect of the present invention relates to the
pyridazinone derivative of the following formula (1):
##STR00004##
[0060] in which
[0061] I) R.sup.1 represents H, C.sub.1-C.sub.5-alkyl,
C.sub.3-C.sub.10-cycloalkyl, aryl, or a side chain residue of all
the natural amino acids,
[0062] II) R.sup.2 represents H, C.sub.1-C.sub.5-alkyl,
C.sub.3-C.sub.10-cycloalkyl, aryl, or a side chain residue of all
the natural amino acids,
[0063] III) R.sup.3 represents H, C.sub.1-C.sub.5-alkyl, aryl,
hydroxy, C.sub.3-C.sub.10-alkoxy, or halogen,
[0064] IV) R.sup.4 represents H, C.sub.1-C.sub.5-alkyl,
C.sub.3-C.sub.10-cycloalkyl, or aryl,
[0065] V) R.sup.5 represents H, C.sub.1-C.sub.5-alkyl,
C.sub.3-C.sub.10-cycloalkyl, or aryl,
[0066] VI) R.sup.6 and R.sup.7 independently of one another each
represent H, C.sub.1-C.sub.5-alkyl, C.sub.3-C.sub.10-cycloalkyl, or
aryl,
[0067] VII) X represents --CH.sub.2OR.sup.9 (R.sup.9 is
C.sub.1-C.sub.5-alkyl, C.sub.3-C.sub.10-cycloalkyl, or aryl),
--CH.sub.2OC(.dbd.O)R.sup.10 (R.sup.10 is C.sub.1-C.sub.5-alkyl,
C.sub.3-C.sub.10-cycloalkyl, or aryl), or --CH.sub.2--W (W is
halogen), or pharmaceutically acceptable salt thereof, which is
useful as an inhibitor for caspase.
[0068] In the compound of formula (1) according to the present
invention, R.sup.1 preferably represents a side chain residue of
all the natural amino acids, more preferably --CH.sub.2 COOH. The
compound of formula (1) may include the two kinds of stereoisomers,
or mixtures thereof (diastereomeric mixtures) when the carbon to
which R.sup.1 is attached becomes a stereocenter due to the R.sup.1
group. The compound of formula (1) may include an ester form (--CO
Y wherein Y.sup.1 is C.sub.1-C.sub.5-alkyl), a sulfonamide form
(--CONHSO Y.sup.2 wherein Y.sup.2 is C.sub.1-C.sub.5-alkyl), and a
pharmaceutically acceptable salt form, when R.sup.1 is a side chain
residue of an amino acid containing carboxyl moiety; or the
compound of formula (1) may also exist in the form of a
pharmaceutically acceptable salt when R.sup.1 is a side chain
residue of an amino acid containing a base moiety.
[0069] The compound of the present invention (formula 1a) may exist
in the form of a cyclic ketal (formula 1b) when R.sup.1 is
--CH.sub.2COOH, and so a skilled artisan may understand that the
cyclic ketal form (formula 1b) may also be covered by the present
invention.
##STR00005##
[0070] Also, the equilibrium forms of said compounds should be
understood to cover their tautomeric forms.
[0071] R.sup.2 preferably represents C.sub.1-C.sub.5-alkyl, more
preferably methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, or
t-butyl. The compound of formula (1) may include the two kinds of
stereoisomers, or mixtures thereof (diastereomeric mixtures) when
the carbon to which R.sup.2 is attached becomes a stereocenter due
to the R.sup.2 group. The compound of formula (1) may include an
ester form (--CO Y.sup.1 wherein Y.sup.1 is C.sub.1-C.sub.5-alkyl),
a sulfonamide form (--CONHSO Y.sup.2 wherein Y.sup.2 is
C.sub.1-C.sub.5-alkyl), and a pharmaceutically acceptable salt
form, when R.sup.2 is a side chain residue of an amino acid
containing carboxyl moiety; or the compound of formula (1) may also
exist in the form of a pharmaceutically acceptable salt when
R.sup.2 is a side chain residue of an amino acid containing a base
moiety.
[0072] R.sup.3 preferably represents H, C.sub.1-C.sub.5-alkyl,
aryl, C.sub.1-C.sub.5-alkoxy, or halogen, more preferably H,
methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, or t-butyl,
methoxy, ethoxy, fluoro, or chloro.
[0073] R.sup.4 preferably represents H.
[0074] R.sup.5 preferably represents C.sub.1-C.sub.5-alkyl
substituted by C.sub.3-C.sub.10-cycloalkyl or aryl, each of which
is substituted or unsubstituted; or represents substituted or
unsubstituted aryl. R.sup.5 more preferably represents
C.sub.1-C.sub.5-alkyl substituted by C.sub.3-C.sub.10-cycloalkyl or
aryl, each of which is unsubstituted or substituted by one or more
substituents selected from the group consisting of
C.sub.1-C.sub.5-alkyl, hydroxy, C.sub.1-C.sub.5-alkoxy and halogen;
or represents aryl which is unsubstituted or substituted by one or
more substituents selected from the group consisting of
C.sub.1-C.sub.5-alkyl, hydroxy, C.sub.1-C.sub.5-alkoxy and halogen.
For example, R.sup.5 is phenyl, naphthyl, indolyl, quinolinyl,
isoquinolyl, imidazolinyl, isoxazolyl, oxazolyl or thiazolyl, or is
methyl substituted by phenyl, naphthyl, indolyl, quinolinyl,
isoquinolyl, imidazolinyl, isoxazolyl, oxazolyl, thiazolyl or
cyclohexyl, each of which is unsubstituted or substituted by one or
more substituents selected from the group consisting of methyl,
ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, methoxy,
ethoxy, trihalomethyl and halogen.
[0075] R.sup.6 and R.sup.7 each preferably represent H.
[0076] R.sup.9 preferably represents aryl substituted by one or
more halogens, more preferably phenyl substituted by one or more
fluorines, and most preferably 2,3,5,6-tetrafluorophenyl.
[0077] R.sup.10 preferably represents aryl substituted by one or
more halogens, more preferably phenyl substituted by one or more
chlorines, most preferably 2,6-dichlorophenyl.
[0078] W preferably represents F.
[0079] The most preferred compounds are these selected from the
following group: [0080]
3-{2-[5-(2-tert-butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-butyrylam-
ino}-5-fluoro-4-oxo-pentanoic acid (1); [0081]
(S)-3-{2-[5-(2-tert-butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-butyr-
ylamino}-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid (2);
[0082]
(S)-3-{2-[5-(2-tert-butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-propi-
onylamino}-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid
(3); [0083]
(S)-3-{2-[5-(2-tert-butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl-
]-acetylamino}-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid
(4); [0084]
(S)-3-{2-[5-(2-tert-butyl-benzyl)-6-oxo-6H-pyridazin-1-yl]-butyryl-
amino}-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid (5);
and [0085]
(S)-3-{2-[3-(2-tert-butyl-benzyl)-6-oxo-6H-pyridazin-1-yl]-butyryl-
amino}-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)-pentanoic acid (6).
[0086] The processes for preparation of the novel pyridazinone
derivative of formula (1) showing an inhibitory activity against
caspases are depicted in the following Reaction Schemes 1 to 3.
However, these illustrated in the following Reaction Schemes
represent only the typical processes used in the present invention.
The manipulation order, reagent, reaction condition, solvent, etc.
may be changed with no limit.
##STR00006##
[0087] In the above Reaction Scheme, R5' represents R5 except for
CH.sub.2 group.
[0088] In Reaction Scheme 1, the aromatic aldehyde and
6-alkyl-4,5-dihydro-2H-pyridazin-3-one are reacted in ethanol in
the presence of a base to give the pyridazinone compound (3). This
compound (3) is reacted with .alpha.-halo-.alpha.-alkylacetate in a
suitable solvent in the presence of a base to give the compound
(4). If necessary, the compound (4) is hydrolyzed to give the
deprotected carboxylic acid derivative (5).
##STR00007##
[0089] In the above Reaction Scheme 2 and the following Reaction
Scheme 3, Z represents --OR.sup.9 (R.sup.9 is
C.sub.1-C.sub.5-alkyl, C.sub.3-C.sub.10-cycloalkyl, or aryl),
--OC(.dbd.O)R.sup.10 (R.sup.10 is C.sub.1-C.sub.5-alkyl,
C.sub.3-C.sub.10-cycloalkyl, or aryl), or --W (W is halogen).
[0090] As depicted in Reaction Scheme 2, the carboxylic acid
derivative (5) is coupled with the aspartic acid derivative (10)
(see the following Reaction Scheme 3) to give the compound (6),
which is then subjected to Dess-Martin periodene oxidation
reaction, and if necessary deprotection reaction, to give the
desired compound (1).
[0091] The functional group Z in the compound (1) of Reaction
Scheme 2 may be formed first by synthesizing the compound (10)
already having the desired Z group according to the process of
Reaction Scheme 3, and by reacting the compound (10) with the
carboxylic acid compound (5) (see WO 00/23421). Or, the desired Z
group may be introduced later according to the process of Reaction
Scheme 3 after the carboxylic acid compound (5) is combined with
the aspartic acid (P-t-Bu) methyl ester and hydrolyzed. When Z is
F, the racemic compound may be prepared according to a method known
in Tetrahedron Letters, 1994, 35(52), 9693-9696.
##STR00008##
[0092] The compound of formula (1) according to the present
invention has a broad spectrum of inhibitory activity against
caspases as demonstrated by the results of the following
Experiments, and so has an effect for preventing inflammation and
apoptosis. Thus, the present invention provides a pharmaceutical
composition for inhibiting caspases, specifically a therapeutic
composition for preventing inflammation and apoptosis, comprising
the compound of formula (1) or pharmaceutically acceptable salt
thereof as an active ingredient together with the pharmaceutically
acceptable carrier. Specifically, the composition of the present
invention has a therapeutic or preventing effect for dementia,
cerebral stroke, brain impairment due to AIDS, diabetes, gastric
ulcer, cerebral injury by hepatitis, hepatitis-induced hepatic
diseases, acute hepatitis, fulminant hepatic failure, sepsis, organ
transplantation rejection, rheumatic arthritis, cardiac cell
apoptosis due to ischemic cardiac diseases, or liver cirrhosis.
[0093] Further, the present invention provides a use of the
compound of formula (1) or pharmaceutically acceptable salt thereof
for inhibiting caspase, specifically for preventing inflammation
and apoptosis. The present invention still further provides a
method for preventing inflammation and apoptosis in a patient,
which comprises administering a therapeutically effective amount of
the compound of formula (1) or pharmaceutically acceptable salt
thereof to the patient. The present invention still further
provides a method for the treatment or prevention of dementia,
cerebral stroke, brain impairment due to AIDS, diabetes, gastric
ulcer, cerebral injury by hepatitis, hepatitis-induced hepatic
diseases, acute hepatitis, fulminant hepatic failure, sepsis, organ
transplantation rejection, rheumatic arthritis, cardiac cell
apoptosis due to ischemic cardiac diseases, or liver cirrhosis in a
patient, which comprises administering a therapeutically effective
amount of the compound of formula (1) or pharmaceutically
acceptable salt thereof to the patient.
[0094] The compound of formula (1) may be formulated into various
pharmaceutical forms for administration purpose. To prepare the
pharmaceutical composition according to the present invention, an
effective amount of the compound of formula (1) or pharmaceutically
acceptable salt thereof is mixed with a pharmaceutically acceptable
carrier that may be selected depending on the formulation to be
prepared.
[0095] The caspase inhibitor compound may be formulated as a
parenteral injection, percutaneous or oral preparation, depending
on its application purpose. It is especially advantageous to
formulate the composition in a unit dosage form for ease of
administration and uniformity of dosage.
[0096] For the oral preparation, any usual pharmaceutical carrier
may be used. For example, water, glycols, oils, alcohols and the
like may be used for such oral liquid preparations as suspensions,
syrups, elixirs and solutions; or starches, sugars, kaolin,
lubricants, binders, disintegrating agents and the like may be used
for such solid preparations as powders, pills, capsules and
tablets. Due to their ease of administration, tablets and capsules
are the most advantageous dosage unit forms. It is also desirable
for tablets and pills to be formulated into enteric-coated
preparation.
[0097] For the parenteral preparation, sterile water is usually
used as the carrier, though other ingredients such as solubility
aids may be used. Injections, for example, sterilized aqueous or
oily suspension for injection, can be prepared according to the
known procedure using suitable dispersing agent, wetting agent, or
suspending agent. Solvents that can be used for preparing
injections include water, Ringer's fluid, and isotonic NaCl
solution, and also sterilized fixing oil may be conveniently used
as the solvent or suspending media. Any non-stimulative fixing oil
including mono- or di-glyceride may be used for this purpose. Fatty
acid such as oleic acid may also be used for injections.
[0098] For the percutaneous administration, the carrier may include
a penetration enhancing agent and/or a suitable wetting agent,
optionally combined with suitable additives having no significant
skin irritation. Said additives may facilitate the administration
through the skin and/or may assist preparation of a desired
composition. These percutaneous preparations are administered via
various manners, e.g., as a transdermal patch, a spot-on, or an
ointment.
[0099] When the caspase inhibitor of the present invention is used
for clinical purpose, it is preferable to administer to the subject
patient in an amount ranging from 0.1 to 100 mg per kg of body
weight a day. The total daily dosage may be administered once or
over several times. However, specific administration dosage for an
individual patient can be varied with specific compound used, body
weight, gender, hygienic condition, or diet of subject patient,
time or method of administration, excretion rate, mixing ratio of
agent, severity of disease to be treated, etc.
MODE FOR INVENTION
[0100] The present invention will be more specifically explained by
the following examples. However, it should be understood that these
examples are intended to illustrate the present invention but not
in any manner to limit the scope of the present invention.
Preparation 1-1
1-Bromomethyl-2-tert-butyl-benzene
[0101] To 1-tert-butyl-2-methyl-benzene (940 mg, 6.34 mmol), NBS
(1.24 g, 1.1 eq) and AIBN (20 mg, catalytic amount) was added
CCl.sub.4 (12 ml), and the mixture was refluxed for 1 h. The
suspended particles were removed by filtration, and washed with
CCl.sub.4. The organic layers were combined, and concentrated under
reduced pressure to give 1.5 g of a yellow liquid in a
stoichiometric yield.
[0102] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 7.46 (m, 1H), 7.38
(m, 1H), 7.22-7.21 (m, 2H), 4.83 (s, 2H), 1.46 (s, 9H)
Preparation 1-2
2-tert-Butyl-benzaldehyde
[0103] To the compound of Preparation 1-1) (1.00 g, 4.4 mmol) were
added NaHCO.sub.3 (1.85 g, 5.0 eq) and DMSO (10 ml), and the
mixture was heated at 100.degree. C. for 30 min. The reaction
mixture was extracted with ethyl acetate (100 ml.times.2), washed
with water (50 ml.times.3) and aqueous sodium chloride solution (50
ml.times.1), dried (anhydrous Na.sub.2SO.sub.4), and concentrated
under reduced pressure. The residue was purified by column
chromatography (5% ethyl acetate-hexane) to give the title compound
(750 mg, Yield 99%).
[0104] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 10.85 (s, 1H),
7.93 (d, 1H), 7.49 (m, 1H), 7.32 (m, 1H), 7.25 (m, 1H), 1.52 (s,
9H)
Preparation 1-3
4-(2-tert-Butyl-benzyl)-6-methyl-2H-pyridazin-3-one
[0105] To the compound of Preparation 1-2) (324 mg, 2.0 mmol) were
added 6-methyl-4,5-dihydro-2H-pyridazin-3-one (Aldrich, 224 mg, 1.0
eq), KOH (168 mg, 3.0 eq) and EtOH (10 ml), and the mixture was
heated under reflux for 18 h. The reaction mixture was neutralized
by 1N aqueous hydrochloric acid solution (3.0 ml), and distilled
under reduced pressure. The residue was dissolved in excess ethyl
acetate (50 ml), washed with aqueous sodium chloride solution,
dried (anhydrous Na.sub.2SO.sub.4), and concentrated under reduced
pressure. The residue was purified by column chromatography (50%
ethyl acetate-hexane) to give the title compound (292 mg, Yield
57%).
[0106] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 12.66 (br s, 1H),
7.48 (d, 1H), 7.26-7.20 (m, 2H), 7.02 (d, 1H), 6.40 (s, 1H), 4.22
(s, 2H), 2.19 (s, 3H), 1.34 (s, 9H)
Preparation 1-4
2-[5-(2-tert-Butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-butyric
acid ethyl ester
[0107] To a mixture of the compound of Preparation 1-3) (90 mg,
0.35 mmol) and Cs.sub.2CO.sub.3 (342 mg, 3.0 eq) were added DMF (7
ml) and 2-bromo-butyric acid ethyl ester (343 mg, 5.0 eq), and the
mixture was stirred under nitrogen gas at room temperature for 3 h.
The reaction mixture was concentrated under reduced pressure, and
the residue was extracted twice with ethyl acetate (100 ml). The
extract was washed with saturated sodium hydrogen carbonate
solution (NaHCO.sub.3, 100 ml.times.2) and aqueous sodium chloride
solution, dried (anhydrous Na.sub.2SO.sub.4), and concentrated
under reduced pressure. The residue was purified by column
chromatography (20% ethyl acetate-hexane) to give the title
compound in a stoichiometric yield.
[0108] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 7.46 (d, 1H),
7.25-7.18 (m, 2H), 7.02 (d, 1H), 6.33 (s, 1H), 5.43 (m, 1H), 4.19
(m, 1H), 4.17 (s, 2H), 2.24 (m, 2H), 2.16 (s, 3H), 1.33 (s, 9H),
1.22 (t, 3H), 0.91 (t, 3H)
Preparation 1-5
2-[5-(2-tert-Butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-butyric
acid
[0109] The compound of Preparation 1-4) (128 mg) was dissolved in a
solvent mixture (6 ml, tetrahydrofuran:MeOH:H.sub.2O=3:2:1),
LiOH.H.sub.2O (29 mg, 2.0 eq) was added thereto, and the mixture
was stirred at room temperature for about 2 h. The reaction mixture
was neutralized by 1N aqueous hydrochloric acid solution, and
distilled under reduced pressure to remove most tetrahydrofuran.
The residue was dissolved in excess ethyl acetate (50 ml), washed
with aqueous sodium chloride solution, dried (anhydrous Na.sub.2
SO.sub.4), and concentrated under reduced pressure to give the
title compound in a stoichiometric yield. This compound was used in
the next reaction without further purification.
Preparation 1-6
3-{2-[5-(2-tert-Butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-butyrylami-
no}-5-fluoro-4-oxo-pentanoic acid tert-butyl ester
[0110] A mixture of the carboxylic acid derivative obtained in
Preparation 1-5) (125 mg, 0.36 mmol),
3-amino-5-fluoro-4-hydroxy-pentanoic acid tert-butyl ester (see:
Tetrahedron Letters, 1994, 35(52), 9693-9696, 83 mg, 1.1 eq) and
HATU (178 mg, 1.3 eq) was cooled to 0.degree. C., triethylamine
(0.20 ml, 4.0 eq) in DMF solvent (5 ml) was added thereto, and the
mixture was reacted at room temperature for 3 h. The solvent was
distilled under reduced pressure. The residue was extracted with
ethyl acetate (30 ml.times.2), washed with water, aqueous sodium
hydrogen carbonate solution and aqueous sodium chloride solution,
dried (anhydrous Na.sub.2SO.sub.4), and concentrated under reduced
pressure. To the compound thus obtained and Des s-Martin reagent
(305 mg, 2.0 eq) was added anhydrous dichloromethane (4 ml), and
the mixture was stirred at room temperature for 1 h. Isopropyl
alcohol (1 ml) was added to stop the reaction. The reaction mixture
was filtered through celite under reduced pressure to remove the
solid, and extracted with ethyl acetate (20 ml.times.2). The
extract was washed with water, saturated sodium hydrogen carbonate
solution and aqueous sodium chloride solution, dried (anhydrous
Na.sub.2SO.sub.4), and concentrated under reduced pressure. The
residue was purified by Prep-TLC (30-40% ethyl acetate-hexane) to
give the title compound (125 mg, Yield 67%).
[0111] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 7.48-7.42 (m, 2H),
7.24 (t, 1H), 7.18 (t, 1H), 6.99 (m, 1H), 6.36 (s, 1H), 5.49 (m,
1H), 5.18-4.90 (m, 2H), 4.83 (m, 1H), 4.17 (s, 2H), 2.98-2.62 (m,
2H), 2.19 (two s, 3H), 2.25-2.12 (m, 2H), 1.39 (two s, 9H), 1.32
(s, 9H), 0.90 (m, 3H)
Example 1
3-{2-[5-(2-tert-Butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-butyrylami-
no}-5-fluoro-4-oxo-pentanoic acid
##STR00009##
[0113] The compound of Preparation 1-6) (120 mg, 0.23 mmol) was
dissolved in dichloromethane (4 ml), and trifluoroacetic acid (2
ml) was added thereto at 0.degree. C. The reaction mixture was
stirred for 1 h while being slowly warmed to room temperature, and
concentrated under reduced pressure. The residue was purified by
Prep-TLC (10% methanol-dichloromethane) to give the title compound
(90 mg, Yield 82%).
[0114] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 7.70 (two br s,
1H), 7.46 (d, 1H), 7.24 (t, 1H), 7.18 (t, 1H), 6.97 (d, 1H), 6.46
& 6.43 (two s, 1H), 5.37 (m, 1H), 5.05-4.70 (m, 3H), 4.14 (s,
2H), 3.18-2.72 (m, 2H), 2.23 (two s, 3H), 2.25-2.15 (m, 2H), 1.32
(s, 9H), 0.92 (m, 3H)
Preparation 2-1
(S)-3-Benzyloxycarbonylamino-4-hydroxy-5-(2,3,5,6-tetrafluoro-phenoxy)-pen-
tanoic acid tert-butyl ester
[0115] To N-benzyloxycarbonyl-.beta.-t-butylaspartic acid (17.93 g,
55.46 mmol) and NMM (6.70 ml, 1.10 eq) was added anhydrous
tetrahydrofuran (150 ml) under nitrogen gas, which was maintained
at -15.degree. C. Isobutylchloroformate (7.56 ml, 1.05 eq) was
added thereto, and reaction mixture was stirred for about 20 min.
The mixture was maintained at 0.degree. C., during which
diazomethane-ether solution (synthesized from 2.0 eq
1-methyl-3-nitro-1-nitroso-guanidine, 60 ml) was added, and stirred
at 0.degree. C. for 30 min to give a diazoketone derivative. 30%
HBr/AcOH (22.6 ml, 2.0 eq) was added thereto at 0.degree. C., and
stirred for 30 min. The reaction mixture was extracted with ethyl
acetate, washed with water, saturated sodium hydrogen carbonate
solution (twice) and aqueous sodium chloride solution, dried
(anhydrous Na.sub.2SO.sub.4), and concentrated under reduced
pressure to give a bromomethylketone derivative (22.2 g) in a
stoichiometric yield.
[0116] The bromomethylketone derivative (22.2 g, 55.45 mmol) and
2,3,5,6-tetrafluorophenol (11.05 g, 1.2 eq) were dissolved in
dimethylformamide (130 ml), KF (8.05 g, 2.5 eq) was added, and the
mixture was stirred at room temperature for 2 h. The reaction
mixture was concentrated under reduced pressure. The residue was
extracted with ethyl acetate, washed with water, saturated sodium
hydrogen carbonate solution (twice) and aqueous sodium chloride
solution, dried (anhydrous Na.sub.2SO.sub.4), and concentrated
under reduced pressure to give
2,3,5,6-tetrafluorophenoxymethylketone derivative. This compound
was dissolved in methanol (150 ml), NaBH.sub.4 (4.19 g, 2.0 eq) was
slowly added thereto at 0.degree. C., and the mixture was stirred
for 1 h. Saturated ammonium acetate solution was added to stop the
reaction, and the reaction mixture was distilled under reduced
pressure to remove methanol. The residue was extracted with ethyl
acetate (200 ml.times.2), washed with water and aqueous sodium
chloride solution, dried (anhydrous Na.sub.2SO.sub.4), and
concentrated under reduced pressure. The residue was
purified-separated by column chromatography (10-20% ethyl
acetate/hexane) to give the title compound (19.6 g, Yield 73%).
Preparation 2-2
(S)-3-Amino-4-hydroxy-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic
acid tert-butyl ester
[0117] The compound of Preparation 2-1) (19.6 g, 40.2 mmol) was
dissolved in MeOH (130 ml), Pd/C (Aldrich, 10%, 1.0 g) was added,
and the mixture was stirred under hydrogen gas for 3 h. The
reaction mixture was filtered through celite to remove Pd/C, and
washed with MeOH. The filtrate was distilled under reduced pressure
to give the title compound (13.17 g, Yield 93%).
[0118] .sup.1H-NMR (400 MHz, DMSO-d.sub.6) .delta. 8.2 (br, 2H),
7.6-7.5 (m, 1H), 5.9 (m, 1H), 4.3-4.1 (m, 3H), 3.6 (m, 1H), 2.7 (m,
1H), 1.4 (s, 9H)
Preparation 2-3
(S)-3-{2-[5-(2-tert-Butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-butyry-
lamino}-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid
tert-butyl ester
[0119] A mixture of the carboxylic acid derivative obtained in
Preparation 1-5) (70 mg, 0.20 mmol), the compound of Preparation
2-2) (79 mg, 1.1 eq) and HATU (99 mg, 1.3 eq) was cooled to
0.degree. C., triethylamine (0.11 ml, 4.0 eq) in DMF solvent (5 ml)
was added thereto, and the mixture was reacted at room temperature
for 1.5 h. The solvent was distilled under reduced pressure. The
residue was extracted with ethyl acetate (30 ml.times.2), wished
with water, aqueous sodium hydrogen carbonate solution and aqueous
sodium chloride solution, dried (anhydrous Na.sub.2SO.sub.4), and
concentrated under reduced pressure. To the compound thus obtained
and Dess-Martin reagent (170 mg, 2.0 eq) was added anhydrous
dichloromethane (4 ml), and the mixture was stirred at room
temperature for 1 h. Isopropyl alcohol (1 ml) was added to stop the
reaction. The reaction mixture was filtered through celite under
reduced pressure to remove the solid, and extracted with ethyl
acetate (20 ml.times.2). The extract was wished with water,
saturated sodium hydrogen carbonate solution and aqueous sodium
chloride solution, dried (anhydrous Na.sub.2SO.sub.4), and
concentrated under reduced pressure. The residue was purified by
Prep-TLC (30% ethyl acetate-hexane) to give the title compound (110
mg, Yield 81%).
[0120] .sup.1H-NMR (500 MHz, CDCl.sub.3) 7.54 (m, 1H), 7.47 (m,
1H), 7.19 (t, 1H), 7.00 (t, 1H), 6.75 (m, 1H), 6.37 (m, 1H), 5.50
(m, 1H), 5.16-4.96 (m, 2H), 4.86 (m, 1H), 4.17 (m, 2H), 3.03-2.61
(m, 2H), 2.20 (two s, 3H), 2.26-2.15 (m, 2H), 1.39 & 1.38 (two
s, 9H), 1.33 (s, 9H), 0.91 (m, 3H)
Example 2
(S)-3-{2-[5-(2-tert-Butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-butyry-
lamino}-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid
##STR00010##
[0122] The compound of Preparation 2-3) (10 mg, 0.15 mmol) was
dissolved in dichloromethane (4 ml), and trifluoroacetic acid (2
ml) was added thereto at 0.degree. C. The reaction mixture was
stirred for 1 h while being slowly warmed to room temperature, and
concentrated under reduced pressure. The residue was purified by
Prep-TLC (65% ethyl acetate-hexane) to give the title compound (85
mg, Yield 91%).
[0123] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 7.75 & 7.55
(two br s, 1H), 7.45 (m, 1H), 7.23 (t, 1H), 7.17 (m, 1H), 6.96 (m,
1H), 6.74 (m, 1H), 6.44 (two s, 1H), 5.43-5.34 (m, 1H), 5.00-4.70
(m, 3H), 4.12 (m, 2H), 3.11 (m, 1H), 2.77 (m, 1H), 2.20 & 2.21
(two s, 3H), 2.26-2.16 (m, 2H), 1.31 & 1.30 (two s, 9H), 0.92
(m, 3H)
Preparation 3-1
2-[5-(2-tert-Butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-propionic
acid ethyl ester
[0124] To a mixture of the compound of Preparation 1-3) (26 mg,
0.10 mmol) and Cs.sub.2CO.sub.3 (65 mg, 2.0 eq) were added DMF (5
ml) and 2-bromo-propionic acid ethyl ester (53 mg, 3.0 eq), and the
mixture was stirred at room temperature under nitrogen gas for 1 h.
The reaction mixture was concentrated under reduced pressure and
the residue was extracted twice with ethyl acetate (100 ml). The
extract was washed with saturated sodium hydrogen carbonate
solution (NaHCO.sub.3, 100 ml.times.2) and aqueous sodium chloride
solution, dried (anhydrous Na.sub.2SO.sub.4), and concentrated
under reduced pressure. The residue was purified by column
chromatography (30% ethyl acetate-hexane) to give the title
compound (30 mg, Yield 84%).
[0125] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 7.46 (d, 1H), 7.23
(t, 1H), 7.18 (t, 1H), 7.00 (d, 1H), 6.33 (s, 1H), 5.55 (qt, 1H),
4.20 (m, 2H), 4.16 (s, 2H), 2.16 (s, 3H), 1.68 (d, 3H), 1.33 (s,
9H), 1.23 (t, 3H)
Preparation 3-2
(S)-3-{2-[5-(2-tert-Butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-propio-
nylamino}-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid
tert-butyl ester
[0126] The compound of Preparation 3-1) (30 mg, 0.084 mmol) was
hydrolyzed according to the same procedure as Preparation 1-5) to
give a carboxylic acid derivative (29 mg, 0.084 mmol). A mixture of
this carboxylic acid derivative, the compound of Preparation 2-2)
(35 mg, 1.1 eq) and HATU (44 mg, 1.3 eq) was cooled to 0.degree.
C., triethylamine (0.05 ml, 4.0 eq) in DMF solvent (5 ml) was added
thereto, and the mixture was reacted at room temperature for 2 h.
The solvent W is distilled under reduced pressure. The residue was
extracted with ethyl acetate (30 ml.times.2), washed with water,
aqueous sodium hydrogen carbonate solution and aqueous sodium
chloride solution, dried (anhydrous Na.sub.2SO.sub.4), and
concentrated under reduced pressure. To the compound thus obtained
and Dess-Martin reagent (76 mg, 2.0 eq) was added anhydrous
dichloromethane (4 ml), and the mixture was stirred at room
temperature for 1 h. Isopropyl alcohol (1 ml) was added to stop the
reaction. The reaction mixture was filtered through celite under
reduced pressure to remove the solid, and extracted with ethyl
acetate (20 ml.times.2). The extract was washed with water,
saturated sodium hydrogen carbonate solution and aqueous sodium
chloride solution, dried (anhydrous Na.sub.2SO.sub.4), and
concentrated under reduced pressure. The residue was purified by
Prep-TLC (40% ethyl acetate-hexane) to give the title compound (35
mg, Yield 60%).
[0127] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 7.47 (d, 1H), 7.37
(t, 1H), 7.24 (t, 1H), 7.18 (t, 1H), 6.99 (d, 1H), 6.73 (m, 1H),
6.37 (two s, 1H), 5.65 (m, 1H), 5.19-4.96 (m, 2H), 4.86 (m, 1H),
4.17 (s, 2H), 3.02-2.62 (m, 2H), 2.19 & 2.18 (two s, 3H), 1.68
(two d, 3H), 1.39 (s, 9H), 1.33 (s, 9H)
Example 3
(S)-3-{2-[5-(2-tert-Butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-propio-
nylamino}-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid
##STR00011##
[0129] The compound of Preparation 3-2) (34 mg, 0.051 mmol) was
dissolved in dichloromethane (4 ml), and trifluoroacetic acid (2
ml) was added thereto at 0.degree. C. The reaction mixture was
stirred for 1 h while being slowly warmed to room temperature, and
concentrated under reduced pressure. The residue was purified by
Prep-TLC (10% methanol/dichloromethane) to give the title compound
(26 mg, Yield 84%).
[0130] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 7.61 (br, 1H),
7.46 (d, 1H), 7.24 (m, 1H), 7.18 (m, 1H), 6.95 (m, 1H), 6.76 (m,
1H), 6.45 (s, 1H), 5.51 (m, 1H), 4.89 (m, 3H), 4.12 (s, 2H),
3.14-2.73 (m, 2H), 2.21 (two s, 3H), 1.67 (two d, 3H), 1.31 (two s,
9H)
Preparation 4-1
[5-(2-tert-Butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-acetic
acid ethyl ester
[0131] To a mixture of the compound of Preparation 1-3) (90 mg,
0.35 mmol) and Cs.sub.2CO.sub.3 (228 mg, 2.0 eq) were added DMF (10
ml) and 2-bromoacetic acid ethyl ester (117 mg, 2.0 eq), and the
mixture was stirred at room temperature under nitrogen gas for 2 h.
The reaction mixture was concentrated under reduced pressure and
the residue was extracted twice with ethyl acetate (100 ml). The
extract was washed with saturated sodium hydrogen carbonate
solution (NaHCO.sub.3, 100 ml.times.2) and aqueous sodium chloride
solution, dried (anhydrous Na.sub.2SO.sub.4), and concentrated
under reduced pressure. The residue was purified by column
chromatography (30% ethyl acetate-hexane) to give the title
compound (104 mg, Yield 87%).
[0132] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 7.46 (d, 1H), 7.23
(t, 1H), 7.19 (t, 1H), 7.01 (d, 1H), 6.35 (s, 1H), 4.87 (s, 2H),
4.24 (qt, 2H), 4.17 (s, 2H), 2.16 (s, 3H), 1.33 (s, 9H), 1.28 (t,
3H)
Preparation 4-2
(S)-3-{2-[5-(2-tert-Butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-acetyl-
amino}-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid
tert-butyl ester
[0133] The compound of Preparation 4-1) (75 mg, 0.22 mmol) was
hydrolyzed according to the same procedure as Preparation 1-5) to
give a carboxylic acid derivative (60 mg, 0.19 mmol, 87%). A
mixture of this carboxylic acid derivative, the compound of
Preparation 2-2) (74 mg, 1.1 eq) and HATU (94 mg, 1.3 eq) was
cooled to 0.degree. C., triethylamine (0.11 ml, 4.0 eq) in DMF
solvent (5 ml) was added thereto, and the mixture was reacted at
room temperature for 2 h. The solvent was distilled under reduced
pressure. The residue was extracted with ethyl acetate (30
ml.times.2), washed with water, aqueous sodium hydrogen carbonate
solution and aqueous sodium chloride solution, dried (anhydrous
Na.sub.2SO.sub.4), and concentrated under reduced pressure. To the
compound thus obtained and Dess-Martin reagent (157 mg, 2.0 eq) was
added anhydrous dichloromethane (4 ml), and the mixture was stirred
at room temperature for 1 h. Isopropyl alcohol (1 ml) was added to
stop the reaction. The reaction mixture was filtered through celite
under reduced pressure to remove the solid, and extracted with
ethyl acetate (20 ml.times.2). The extract was washed with water,
saturated sodium hydrogen carbonate solution and aqueous sodium
chloride solution, dried (anhydrous Na.sub.2SO.sub.4), and
concentrated under reduced pressure. The residue was purified by
Prep-TLC (40% ethyl acetate-hexane) to give the title compound (105
mg, Yield 80%).
[0134] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 7.47 (d, 1H), 7.32
(d, 1H), 7.25 (t, 1H), 7.19 (t, 1H), 6.99 (d, 1H), 6.74 (m, 1H),
6.40 (s, 1H), 5.24-5.03 (m, 2H), 4.91 (m, 1H), 4.85 (s, 2H), 4.16
(two s, 2H), 3.04-2.68 (m, 2H), 2.18 (s, 3H), 1.41 (s, 9H), 1.33
(s, 9H)
Example 4
(S)-3-{2-[5-(2-tert-Butyl-benzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-acetyl-
amino}-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid
##STR00012##
[0136] The compound of Preparation 4-2) (100 mg, 0.15 mmol) was
dissolved in dichloromethane (4 ml), and trifluoroacetic acid (2
ml) was added thereto at 0.degree. C. The reaction mixture was
stirred for 1 h while being slowly warmed to room temperature, and
concentrated under reduced pressure. The residue was purified by
Prep-TLC (65% ethyl acetate/hexane) to give the title compound (59
mg, Yield 67%).
[0137] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 7.71 (br, 1H),
7.45 (d, 1H), 7.23 (t, 1H), 7.17 (t, 1H), 6.95 (d, 1H), 6.75 (m,
1H), 6.46 (s, 1H), 5.06-4.82 (m, 5H), 4.11 (s, 2H), 3.19-2.81 (m,
2H), 2.20 (s, 3H), 1.31 (s, 9H)
Preparation 5-1
4-(2-tert-Butyl-benzyl)-2H-pyridazin-3-one and
6-(2-tert-Butyl-benzyl)-2H-pyridazin-3-one
[0138] To 4,5-dihydro-2H-pyridazin-3-one (192 mg, 1.95 mmol)
obtained by a process known in J. Amer. Chem. Soc., 1945, 67, 60-62
and J. Org. Chem., 1961, 26, 1854-1856, 2-tert-butyl-benzaldehyde
(316 mg, 1.0 eq) obtained in Preparation 1-2) and KOH (220 mg, 2.0
eq) was added EtOH (30 ml), and the mixture was heated under reflux
for 6 h. The reaction mixture was neutralized by 1N aqueous
hydrochloric acid solution, and distilled under reduced pressure to
remove most tetrahydrofuran. The residue was dissolved in excess
ethyl acetate (50 ml), washed with aqueous sodium chloride
solution, dried (anhydrous Na.sub.2SO.sub.4), and concentrated
under reduced pressure. The residue was purified by column
chromatography (50% ethyl acetate-hexane, 10%
methanol/dichloromethane) to give the title compounds
4-(2-tert-butyl-benzyl)-2H-pyridazin-3-one (76 mg) and
6-(2-tert-butyl-benzyl)-2H-pyridazin-3-one (167 mg).
4-(2-tert-Butyl-benzyl)-2H-pyridazin-3-one;
[0139] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 11.73 (s, 1H),
7.65 (d, 1H), 7.47 (d, 1H), 7.24 (t, 1H), 7.20 (t, 2H), 7.01 (d,
1H), 6.50 (m, 1H), 4.21 (s, 2H), 1.34 (s, 9H)
6-(2-tert-Butyl-benzyl)-2H-pyridazin-3-one;
[0140] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 10.60 (s, 1H),
7.61 (s, 1H), 7.45 (d, 1H), 7.25 (t, 1H), 7.18 (t, 2H), 6.97 (d,
1H), 6.44 (s, 1H), 4.15 (s, 2H), 1.40 (s, 9H)
Preparation 5-2
2-[5-(2-tert-Butyl-benzyl)-6-oxo-6H-pyridazin-1-yl]-butyric acid
ethyl ester
[0141] To a mixture of 4-(2-tert-butyl-benzyl)-2H-pyridazin-3-one
obtained in Preparation 5-1) (76 mg, 0.314 mmol) and
Cs.sub.2CO.sub.3 (307 mg, 3.0 eq) were added DMF (4 ml) and
2-bromobutyric acid ethyl ester (306 mg, 5.0 eq), and the mixture
was stirred at room temperature under nitrogen gas for 2 h. The
reaction mixture was concentrated under reduced pressure and the
residue was extracted twice with ethyl acetate (100 ml). The
extract was washed with saturated sodium hydrogen carbonate
solution (NaHCO.sub.3, 100 ml.times.2) and aqueous sodium chloride
solution, dried (anhydrous Na.sub.2SO.sub.4), and concentrated
under reduced pressure. The residue was purified by column
chromatography (10-20% ethyl acetate-hexane) to give the title
compound (100 mg, Yield 89%).
[0142] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 7.69 (d, 1H), 7.51
(d, 1H), 7.30-7.22 (m, 2H), 7.07 (d, 1H), 6.50 (m, 1H), 5.56 (dd,
1H), 4.25 (m, 4H), 2.35-2.21 (m, 2H), 1.38 (s, 9H), 1.28 (t, 3H),
0.98 (m, 3H)
Preparation 5-3
(S)-3-{2-[5-(2-tert-Butyl-benzyl)-6-oxo-6H-pyridazin-1-yl]-butyrylamino}-4-
-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid tert-butyl
ester
[0143] The compound of Preparation 5-2) (94 mg, 0.263 mmol) was
hydrolyzed according to the same procedure as Preparation 1-5) to
give a carboxylic acid derivative (86 mg, 0.263 mmol, 100%). A
mixture of this carboxylic acid derivative, the compound of
Preparation 2-2) (102 mg, 1.1 eq) and HATU (130 mg, 1.3 eq) was
cooled to 0.degree. C., triethylamine (0.15 ml, 4.0 eq) in DMF
solvent (5 ml) was added thereto, and the mixture was reacted at
room temperature for 2 h. The solvent W is distilled under reduced
pressure. The residue was extracted with ethyl acetate (30
ml.times.2), washed with water, aqueous sodium hydrogen carbonate
solution and aqueous sodium chloride solution, dried (anhydrous
Na.sub.2SO.sub.4), and concentrated under reduced pressure. To the
compound thus obtained and Dess-Martin reagent (223 mg, 2.0 eq) was
added anhydrous dichloromethane (4 ml), and the mixture was stirred
at room temperature for 1 h. Isopropyl alcohol (1 ml) was added to
stop the reaction. The reaction mixture was filtered through celite
under reduced pressure to remove the solid, and extracted with
ethyl acetate (20 ml.times.2). The extract was washed with water,
saturated sodium hydrogen carbonate solution and aqueous sodium
chloride solution, dried (anhydrous Na.sub.2SO.sub.4), and
concentrated under reduced pressure. The residue was purified by
column chromatography (20-30% ethyl acetate-hexane) to give the
title compound (150 mg, Yield 86%).
[0144] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 7.70 (m, 1H), 7.46
(d, 1H), 7.34 (m, 1H), 7.24 (t, 1H), 7.18 (t, 1H), 7.00 (m, 1H),
6.75 (m, 1H), 6.49 (m, 1H), 5.51 (m, 1H), 5.18-4.94 (m, 2H), 4.87
(m, 1H), 4.18 (m, 2H), 3.02-2.64 (m, 2H), 2.28-2.15 (m, 2H), 1.39
(two s, 9H), 1.32 (s, 9H), 0.92 (m, 3H)
Example 5
(S)-3-{2-[5-(2-tert-Butyl-benzyl)-6-oxo-6H-pyridazin-1-yl]-butyrylamino}-4-
-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid
##STR00013##
[0146] The compound of Preparation 5-3) (146 mg, 0.221 mmol) was
dissolved in dichloromethane (4 ml), and trifluoroacetic acid (2
ml) was added thereto at 0.degree. C. The reaction mixture was
stirred for 1 h while being slowly warmed to room temperature, and
concentrated under reduced pressure. The residue was purified by
Prep-TLC (65% ethyl acetate/hexane) to give the title compound (116
mg, Yield 67%).
[0147] .sup.1H-NMR (500 MHz, CDCl.sub.3) 7.80 (m, 2H), 7.45 (d,
1H), 7.24 (m, 1H), 7.18 (m, 1H), 6.96 (m, 1H), 6.76 (m, 1H), 6.57
(m, 1H), 5.41-5.05 (m, 2H), 4.91 (m, 1H), 4.40 (m, 1H), 4.15 (s,
2H), 3.25-2.64 (m, 2H), 2.22 (m, 2H), 1.30 (two s, 9H), 0.94 (m,
3H)
Preparation 6-1
2-[3-(2-tert-Butyl-benzyl)-6-oxo-6H-pyridazin-1-yl]-butyric acid
ethyl ester
[0148] To a mixture of 6-(2-tert-butyl-benzyl)-2H-pyridazin-3-one
obtained in Preparation 5-1) (167 mg, 0.689 mmol) and
Cs.sub.2CO.sub.3 (673 mg, 3.0 eq) were added DMF (4 ml) and
2-bromobutyric acid ethyl ester (672 mg, 5.0 eq), and the mixture
was stirred at room temperature under nitrogen gas for 2 h. The
reaction mixture was concentrated under reduced pressure and the
residue was extracted twice with ethyl acetate (100 ml). The
extract was washed with saturated sodium hydrogen carbonate
solution (NaHCO.sub.3, 100 ml.times.2) and aqueous sodium chloride
solution, dried (anhydrous Na.sub.2SO.sub.4), and concentrated
under reduced pressure. The residue was purified by column
chromatography (20% ethyl acetate-hexane) to give the title
compound (189 mg, Yield 77%).
[0149] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 7.70 (d, 1H), 7.50
(d, 1H), 7.27 (t, 1H), 7.21 (t, 1H), 7.04 (d, 1H), 6.49 (d, 1H),
5.46 (dd, 1H), 4.25-4.19 (m, 4H), 2.31-2.15 (m, 2H), 1.43 (s, 9H),
1.27 (t, 3H), 0.93 (m, 3H)
Preparation 6-2
(S)-3-{2-[3-(2-tert-Butyl-benzyl)-6-oxo-6H-pyridazin-1-yl]-butyrylamino}-4-
-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid tert-butyl
ester
[0150] The compound of Preparation 6-1) (185 mg, 0.519 mmol) was
hydrolyzed according to the same procedure as Preparation 1-5) to
give a carboxylic acid derivative (166 mg, 98%). A mixture of this
carboxylic acid derivative (87 mg, 0.263 mmol), the compound of
Preparation 2-2) (102 mg, 1.1 eq) and HATU (130 mg, 1.3 eq) was
cooled to 0.degree. C., triethylamine (0.15 ml, 4.0 eq) in DMF
solvent (5 ml) was added thereto, and the mixture was reacted at
room temperature for 2 h. The solvent was distilled under reduced
pressure. The residue was extracted with ethyl acetate (30
ml.times.2), washed with water, aqueous sodium hydrogen carbonate
solution and aqueous sodium chloride solution, dried (anhydrous
Na.sub.2SO.sub.4), and concentrated under reduced pressure. To the
compound thus obtained and Dess-Martin reagent (223 mg, 2.0 eq) was
added anhydrous dichloromethane (4 ml), and the mixture was stirred
at room temperature for 1 h. Isopropyl alcohol (1 ml) was added to
stop the reaction. The reaction mixture was filtered through celite
under reduced pressure to remove the solid, and extracted with
ethyl acetate (20 ml.times.2). The extract was washed with water,
saturated sodium hydrogen carbonate solution and aqueous sodium
chloride solution, dried (anhydrous Na.sub.2SO.sub.4), and
concentrated under reduced pressure. The residue was purified by
column chromatography (25-30% ethyl acetate-hexane) to give the
title compound (150 mg, Yield 86%).
[0151] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 7.71 (d, 1H), 7.45
(d, 1H), 7.30 (t, 1H), 7.22 (t, 1H), 7.16 (m, 1H), 6.97 (d, 1H),
6.75 (m, 1H), 6.46 (d, 1H), 5.36 (m, 1H), 5.14-4.95 (m, 2H), 4.85
(m, 1H), 4.15 (m, 2H), 3.00-2.63 (m, 2H), 2.26-2.12 (m, 2H), 1.39
(three s, 18H), 0.90 (m, 3H)
Example 6
(S)-3-{2-[3-(2-tert-Butyl-benzyl)-6-oxo-6H-pyridazin-1-yl]-butyrylamino}-4-
-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid
##STR00014##
[0153] The compound of Preparation 6-2) (142 mg, 0.215 mmol) was
dissolved in dichloromethane (4 ml), and trifluoroacetic acid (2
ml) was added thereto at 0.degree. C. The reaction mixture was
stirred for 1 h while being slowly warmed to room temperature, and
concentrated under reduced pressure. The residue was purified by
Prep-TLC (65% ethyl acetate/hexane) to give the title compound (111
mg, Yield 85%).
[0154] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 7.77 (d, 1H), 7.60
(br s, 1H), 7.45 (d, 1H), 7.22 (t, 1H), 7.16 (t, 1H), 6.95 (d, 1H),
6.76 (m, 1H), 6.51 (s, 1H), 5.28 (m, 1H), 5.05-4.40 (br s, 2H),
4.87 (m, 1H), 4.18 (m, 2H), 3.10-2.68 (m, 2H), 2.24-2.12 (m, 2H),
1.37 (two s, 18H), 0.91 (m, 3H)
[0155] Experiment 1
[0156] Assay for the Caspase Inhibitory Effect
[0157] Caspase-1 and caspase-8 known as cysteine proteases in the
form of .alpha..sub.2.beta..sub.2 were expressed, purified, and
activated by modifying a method known in Thornberry, N. A. et al,
Nature, 1992, 356, 768; Thornberry, N. A. Methods in Enzymology,
1994, 244, 615; Walker, N. P. C. et al. Cell, 1994, 78, 343, and
caspase-9 was also purified by a similar method, and the inhibitory
activity against them was tested. Briefly describing, p10 and p20
subunits (Thornberry, N. A. et al, Nature, 1992, 356, 768) were
expressed in E. coli and purified by nickel column and anionic
exchange chromatography to give caspase-1, caspase-8 and caspase-9.
The fluorescent substrates AcYVAD-AFC for thus obtained caspase-1,
AcDEVD-AFC for caspase-8, and AcLEHD-AFC for caspase-9, were used
for determining specific activity of the synthesized inhibitors.
The enzyme reaction was carried out at 25.degree. C. with various
concentrations of the inhibitors in a buffer solution containing 50
mM HEPES (pH 7.50), 10% (w/v) sucrose, 0.1% (w/v) CHAPS, 100 mM
NaCl, 1 mM EDTA, and 10 mM DTT in the presence of 50 .mu.M
AcYVAD-AFC for 10 nM caspase-1, 50 .mu.M AcDEVD-AFC for 2.1 nM
caspase-8, and 150 .mu.M AcLEHD-AFC for 200 nM caspase-9. The
inhibitory constants K.sub.i and K.sub.obs of the inhibitors were
determined by measuring the reaction velocity with the time lapse
using a fluorescent spectrometer and by obtaining the initial rate
constant. K.sub.i was calculated from the Lineweaver Burk Plot, and
K.sub.obs from the following Equation 1.
K.sub.obs=-ln(1-A.sub.t/A.sub.oo)/t [Equation 1]
[0158] in which
[0159] A.sub.t means cleavage rate (%) at time t, and
[0160] A.sub.oo means the maximum cleavage rate (%).
[0161] Spectra MAX GeminiXS Fluorescent Spectrometer of Molecular
Device Co. was used at the excitation wavelength of 405 nm and the
emission wavelength of 505 nm.
[0162] The in vivo inhibitory activity of the inhibitors was
determined by subjecting Jurkat cell (ATCC TIB-152) to apoptosis
using Fas antibody (Upstate Biotech 05-201) and by detecting the
color change according to the WST-1 method known in Francoeur A. M.
and Assalian A. (1996) Biochemica 3, 19-25 to observe the amount of
alive Jurkat cells when the cells were treated by the inhibitor.
Spectra MAX 340 Spectrometer of Molecular Device Co. was used at
the absorbance wavelength of 440 nm.
TABLE-US-00001 TABLE 1 Caspase-8 K.sub.obs/[I] Jurkat Cell Example
No. (M.sup.-1min.sup.-1) IC.sub.50 (.mu.M) 1 5.5 E6 0.14 2 2.0 E6
0.33 3 4.0 E5 4 2.0 E5 5 2.0 E6 0.17 6 1.7 E5
[0163] Experiment 2
[0164] Therapeutic Effect for Liver Injury Induced by Fas Antibody
in Mouse
[0165] Step 1) Preparation of Blood Sample
[0166] Male Balb/c mice (6 weeks, Charles River Laboratory, Osaka,
Japan) were kept under the conditions of 22.degree. C., 55% of
relative humidity, and light-darkness cycle of 12 hours. Food and
water were supplied ad libitum. In pyrogen-free phosphate buffer
was dissolved the Fas antibody (Jo2; BD pharmingen, San Diego,
Calif.), which was then injected to each mouse in the amount of
0.15 mg/kg through the vein of tail. Immediately after the
injection of the Fas antibody, vehicle (a mixture of
PEG400:ethanol=2:1 was 20-fold diluted with phosphate buffer)
wherein the test compound is dissolved or the vehicle alone was
orally administered to the mice. After 6 hours from the drug
administration, blood samples were obtained from their hearts.
[0167] Step 2: Assay for the Activity of Plasma
Aminotransferase
[0168] The plasma ALT activity was determined for the blood samples
obtained in Step 1 using ALT assay kit (Asan Pharm. Co., Seoul,
Korea) according to the manufacturer's instruction. The results
appeared that the injection of the Fas antibody sharply increases
the ALT activity in plasma, and the test compounds inhibit the
increased enzyme activity in a dose-dependent manner. Based on
these results, ED.sub.50 values of the test compounds were
calculated using Prism software of GraphPad Co. to give 0.001-10
mg/kg.
INDUSTRIAL APPLICABILITY
[0169] As the above results of Experiments show, the compound of
formula (1) of the present invention has an excellent inhibitory
activity against caspase, and particularly exhibits a therapeutic
effect in the animal model of liver injury induced by the Fas
antibody. Therefore, the compound of formula (1) can be
advantageously used for the treatment or prevention of various
diseases and symptoms mediated by caspase.
* * * * *